Supplementary methods:Immunohistochemistry methods used in Anthony et al.

Fluorescent immunostaining, Image Capture, Data collection and Data Analysis varied depending on the used method (Arechavala-Gomeza1, Taylor2 or Beekman3).

Fluorescent immunostaining protocol (All protocols)

  1. Sections were air-dried at room temperature for 20-30 minutes and circled with a hydrophobic peroxidase-antiperoxidase (PAP) pen.
  2. Primary dystrophin (rabbit C-terminal ab15277, Abcam, MA, USA) and spectrin (monoclonal NCL-SPEC1, Leica Microsystems Inc., IL, USA) antibodies were diluted (1:400 and 1:100, respectively) in phosphate buffered saline (PBS) and incubated with the sections for 1 hour at room temperature.
  3. Sections were washed (3x) in PBS for three minutes each.
  4. Each laboratory used secondary antibodies compatible with their microscope e.g. Alexa Fluor® 488 goat anti-mouse IgG (A11017, Molecular Probes, OR, USA) and Alexa Fluor® 568 goat anti-rabbit IgG (A11036, Molecular Probes). These were diluted 1:500 in PBS and incubated for 30 min in the dark at room temperature.
  5. Sections were washed (3x) in PBS for three minutes.
  6. Slides were mounted using anti-fade mounting agent e.g. ProLong® Gold anti-fade reagent (Molecular Probes).

Image acquisition protocol using an epi-fluorescence microscope (Arechavala-Gomeza1 protocol)

  1. Use the control of EACH of the antibodies (dystrophin or spectrin) to set the exposure conditions:
  • Capture an image of the control with the autoexposure at around 100 ms
  • Once the acquisition is finished, uncheck the autoscale and write down the low and high values of the image scaling, as well as the exposure time.
  • Use these values to acquire the control and patient’s images for that antibody
  1. Take at least 4 images per sample (4 control images, 4 for each of the patients)
  2. Save the files in the original grayscale format and label them as follows:

CONTROL/DMD Biopsy number Antibody image number

Eg: CONTROL XXXX Dys-4

DMD XXXX SP-3

Image acquisition (Taylor2 method)

As published in Taylor et al, Neuropathol Appl Neurobiol. 2012 Oct;38(6):591-601.

Images were obtained on a Zeiss 510 Meta confocal microscope. Fields for fluorescent imaging were randomly selected while viewing the spectrin signal under 20X magnification. In the only step involving operator discretion, all image parameters including pinhole size, detector gain, amplifier offset, amplifier gain, and laser intensity were first set for the dystrophin and spectrin channels using normal control tissue, and the same setting used for all samples imaged on a given day. Frame size, scan speed and averaging were the same for all images. For each sample four non-overlapping images for each channel were acquired and stored as 12-bit fluorescent images (.TIFF) for analysis. A single technician (LET) performed all sectioning, staining, and image acquisition steps.

Image acquisition protocol using a Zeiss confocal microscope (Beekman3 protocol)

  1. Use the control of EACH of the antibodies (dystrophin or spectrin) to set the imaging conditions:
  • Using the 25x oil objective, capture an image of the healthy control sample. Use the following settings and adjust the gain as required to acquire a few saturated pixels in the image.

Dystrophin channel / Spectrin channel
-594nm laser intensity: 10 % / -488nm laser intensity: 1,5 %
-Detection window: 599-797 nm / -Detection window: 493-589 nm
-Gain: ~700 (digital gain: 1,0 ) / -Gain: ~700 (digital gain: 1,0 )
-Digital offset: 0 / -Digital offset: 0
-Pinhole: 46,4 / -Pinhole: 50,6

(settings given for sections stained using the Flanigan method);

  • Once the acquisition is finished, write down the above settings.
  • Use these values to acquire the control and patient’s images for that antibody (re-use the first control image to copy the settings automatically)
  1. Take at least 4 images per sample (4 control images, 4 for each of the patients)
  2. Save the files in the original grayscale LSM format and label them as follows:

SubjectID_ab15277-spec_25x_1_1 (slide 1_image 1)

Image analysis (Arechavala-Gomeza1)protocol

*This protocol refers specifically to the use of Metamorph analysis software (Molecular Devices, USA). Some laboratories have updated this protocol to be used with other software, like image J and Image Pro (Media Cybernetics, USA)

Once all images are captured (4 images per staining), look first preferentially at spectrin and control.

  • Open file
  • Select >MeasureRegion measurements
  • Configure: Make sure the following labels are ticked: region label; image name; and all the intensity options (particularly min and max intensity)
  • Use “region toolbar”. Check on the region tool bar: Lock region size (30X30 pixels, for example)
  • Click 10 times in the image at random (this creates 10 small regions of interest (ROI) in the image)
  • Move ROI(circles or whatever you like) to the nearest fibres (1 ROI per fibre)
  • All the numbers are collected in the “region measurements” table. Look at values in order to understand if the selected regions are not in the wanted places. Check for anything odd: a very low value or a very high one (max is 4096), this may mean one of your ROIs is in the wrong place (empty space or speck of antibody).
  • Open log
  • Sheet 1 Excel =>log data (all the data are copied in the excel file) it is important to have labelled your images as detailed above, as it will make them easier to analyse.
  • Press “Control+ arrow (->)”to open the following image
  • Move circles to the appropriate place on the fibres. Scan maximum intensity in order to see if everything is ok
  • When all the controls are analyzed, change folder and carry on with analysis using “control+->”.
  • In case of negative sample, or patient, it is difficult to visualize fibres. Help to contrast fibres with brightness/contrast and move circles to the edge of the fibres THIS WILL NOT ALTER THE VALUES.
  • All the data will be collected in the same excel sheet.
  • Once all the samples’ data is logged, Save the excel file for data analysis.

Image Analysis (Taylor2 method)

Image analysis was performed in the Metamorph (Molecular Devices, Inc) software program using a custom script. The steps involved in the analysis are outlined below (and represented in Fig. 1):

1.The original spectrin and dystrophin images are opened in Metamorph

2.An intensity threshold for the spectrin signal is chosen by the user, and recorded in a notebook for each image. Pixels with signals above this threshold constitute spectrin regions of interest

3.Regions of interest are converted into a binary mask where areas of interest have a pixel intensity value of 1 and background has a pixel intensity value of 0

4.In order to eliminate background noise, spectrin areas smaller than 3x3 contiguous pixels are removed from the image by an erosion step, leaving areas of equal to or greater than 3x3 contiguous pixels that are presumed to define contiguous muscle membrane.

5.The resulting image is dilated to restore positive regions

6.The dilated image is used to create a spectrin mask

7.The spectrin mask is overlaid on to the spectrin image and intensities and areas are quantified

8.The spectrin mask is overlaid onto the dystrophin image and intensities and areas are quantified

9.All data is recorded into a spreadsheet and saved by the user

The Excel file contains values for average dystrophin intensity, average spectrin intensity, spectrin area, and a dystrophin/spectrin ratio for each image pair analyzed. Dystrophin intensity is recorded for all contiguous spectrin positive regions and summed to create a total dystrophin intensity value for a single image. Areas of contiguous spectrin positive regions are summed to give a total spectrin positive area, and the total dystrophin intensity value then divided by the total area of spectrin positive regions, with the result defined as the average dystrophin intensity for the whole image. Spectrin intensity is recorded for all contiguous spectrin positive regions and summed to create a total intensity value for a single image. The total spectrin intensity value is then divided by the total area of spectrin positive regions, and the result defined as the average spectrin intensity for the whole image. Average dystrophin intensity was divided by average spectrin intensity to give a dystrophin/spectrin ratio. These values were recorded for each of the four images per sample. The four dystrophin intensity values and dystrophin/spectrin ratio values were themselves averaged to generate an overall dystrophin intensity and dystrophin/spectrin ratio for each sample.

Image analysis (Beekman protocol)

*This protocol usesDefiniens image analysis software for actual analysis and ImageJ software to convert the LSM confocal fluorescent images to a TIFF format that can be processed by Definiens.

Image analysis was performed using Definiens Architect software (version 2.0) with a customized algorithm and application. Using the spectrin signal, the software detects the individual muscle fibres and identifies for each fiber the sarcolemma and cytoplasm. The intensity of the dystrophin signal at the sarcolemma is considered to be dystrophin protein properly localized to the membrane. The software measures the dystrophin intensity at the sarcolemmal membrane per muscle fibre. The mean dystrophin membrane intensity is the average intensity of all pixels present in the spectrin-positivemembrane area of an individual fibre. In addition to the mean intensity, minimum, maximum, and quartiledystrophin intensity values are determined per fibre. For the samples described in this study; 4 images/section, on average ~350 fibres per section were analysed. These 350 fibre values are used to calculate thedystrophin mean fiber intensity per section and can also be depicted as a histogram (dystrophin intensity versus number of fibres that have that intensity), to assess the distribution of the dystrophin intensity in the fibre population.The software also calculates morphological parameters of the fibres such as membrane thickness and fibre surface area.

Procedure:

  • Using an ImageJ macro, the confocal 2-layer LSM images are split into two 12-bit TIFF images and saved into a folder with the same name as the original image (spectrin image with extension ‘-001’; dystrophin image with extension ‘-002’).
  • In Definiens Architect software a workspace is started.
  • Pre- Processing of images: The images are loaded by an operator into the workspace (‘one scene per folder’), and sent for ’pre-processing’analysis, for the software to detect the individual muscle fibres (including sarcolemma and cytoplasm) which takesapproximately a minute per image.
  • The pre-processing result is checked visually by an operator;anyincorrectly selected or missed fibre,can be marked by the operator and re-processed by the software.
  • Intensity measurement of images: the images are then processed by the software, to calculate the dystrophin intensity (and morphological) measurements per individualfibre. This process step takes 5 minutes per image to generate CSV files with 45 measurements per fiber.
  • Using an Excel macro averages per section are calculated for all relevant parameters and histograms plotted.

1.Arechavala-Gomeza V, Kinali M, Feng L, et al. Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression. Neuropathol Appl Neurobiol 2010;36:265-274.

2.Taylor LE, Kaminoh YJ, Rodesch CK, Flanigan KM. Quantification of Dystrophin Immunofluorescence in Dystrophinopathy Muscle Specimens. Neuropathol Appl Neurobiol 2012.

3.Beekman C, Testerink J, Giannakopoulos S, et al. P.13.12 An objective method for immunofluorescence analysis of dystrophin levels in muscle from DMD patients in clinical studies. Neuromuscular Disorders 2013;23:812.